KCNJ9 Subcloning Project

Current Status

On step 12 (awaiting sequencing of the "end" of the protein coding sequence)

Protocol

Locate plasmids in SnapGene Files

1. A plasmid map of KCNJ9 in the pGHE backbone was created using the TAAR1_pGHE plasmid and the coding sequence for KCNJ9 from DNASU. The coding sequence for KCNJ9 simply replaced the TAAR1 protein coding sequence in pGHE. A map was also created for KCNJ9 with a stop codon (KCNJ9stp) in the pENTR plasmid (Stock plasmid vector from DNASU).

2. Primers were designed from these plasmid maps, eight total. A KCNJ9 forward HiFi primer ~25nt in length and a KCNJ9 reverse HiFi primer of similar length, resulting in amplification of the KCNJ9 coding sequence for pGHE. For amplification of the pGHE backbone for KCNJ9, a KCNJ9_pGHE reverse primer ~25nt in length and a KCNJ9_pGHE forward primer of similar length. These primers should be arranged for a ~10-15bp overlap and will overlap in pairs like so: KCNJ9 forward/KCNJ9_pGHE reverse, and KCNJ9 reverse/KCNJ9_pGHE forward. Protocol to help with this found here at the top of the page.

3. Order the protein coding sequence in a plasmid of choice from DNASU and primers from New England BioLabs.

4. Send the primers and plasmids, once received from DNASU to GeneWiz for sequencing. Samples will be prepared according to the instructions provided by GeneWiz. (Image below instructions)

5. Once the sequences are confirmed, PCR can be run.

6. Two PCR reactions will be ran, each using one set of primers from step 2 (KCNJ9_pGHE forward and reverse/ KCNJ9 forward and reverse). These reaction mixtures can be made and run at the same time. The first reaction mixture will amplify the pGHE backbone and will contain the source of pGHE backbone (TAAR1_pGHE or B2AR_pGHE) and the primers of this backbone that are specific for KCNJ9, designed in step 2 (KCNJ9_pGHE forward and reverse). The second reaction mixture will amplify the KCNJ9 coding sequence and will contain the KCNJ9 DNA template (purchased from DNASU, step 3), and the primers for this coding sequence in pGHE designed in step 2. PCR protocol found here.

7. PCR products are then analyzed through gel electrophoresis or by the GeneWiz company. Protocol for running and analyzing a gel can be found here.

8. Phosphorylation and ligation of two PCR products into circular DNA. Protocol found here.

9. Circular DNA product then multiplied through transformation by JM109 E. Coli bacterial cells. Protocol found here.

10. If colonies form from bacterial transformation, single colony inoculation is ran. This must be coordinated for a mini-. midi-, or maxi-prep and will define the total volume of the reaction mixture to be inoculated. The goal of this process is to now grow the bacteria in a liquid media rather than a solid agar plate media to facilitate the extraction of DNA after. See protocol for single colony inoculation for details.

11. Inoculation products will then have DNA extracted and isolated through a phenol-chloroform extraction and ethanol precipitation of the DNA. The goal of this process is to obtain a DNA "pellet" in the bottom of the reaction tube following centrifugation. This process will only vary by volume for a mini-, midi-, or maxi-prep. See DNA extraction and precipitation protocol for more details.

12. DNA is then linearized from its previous circular form to facilitate transcription into RNA. Linearization protocol found here.

13. Linearized DNA is transcribed in vitro to RNA. Protocol will be found here. This RNA product can then be stored in the -80 degree freezer downstairs until it is ready for injection into an oocyte.

KCNJ9 Hifi Transformation.